Preparation of DEN virus stocks

DEN virus stocks were prepared by infecting C6/36 cells at low
multiplicity for two weeks with media replacement at one week. The
resultant infected cell culture supernatant was used as the source of
DEN viruses. Viral titers were determined by plaque assay on LLCMK2
cells [42,43].

Construction of rAd-C and rAd-Bg viruses

The design and construction of the bivalent gene, EDIII-4/2, have been described previously [43].
This gene was cloned into plasmid pVAX1 and verified to encode the
predicted ~28 kDa bivalent protein in a rabbit reticulocyte expression
system (TNT system from Promega). The 3' end of this chimeric gene was fused in-frame with the 5' end of the GFP gene
and cloned into the Ad shuttle plasmid pShuttle-CMV. The EDIII-4/2-GFP
expression cassette was then inserted into the E1 region of the Ad5
genome of plasmid pAdEasy-1 by in vivo recombination in E. coli BJ5183 [51]. The resultant rAd plasmid was digested with Pac I
to eliminate plasmid sequences and transfected into HEK 293 cells to
rescue the rAd-Bg virus. The rAd-C virus was constructed in parallel
using a similar strategy except that empty pShuttle-CMV plasmid was
used during in vivo recombination in E. coli.
Recombinant viruses were amplified, purified on CsCl gradients,
dialyzed against 1× PBS containing 10% glycerol, sterilized using
syringe filters and titrated on HEK 293 cells. Both viruses yielded
fairly comparable titers, within 3–4 folds of each other.

Detection of transgene expression in rAd-Bg virus-infected cells

HEK 293 cells were infected at ~25 PFU/cell, for ~24 hours. The
expression of the GFP tag was detected by direct fluorescence
microscopy. The DEN-2 and DEN-4 EDIII components of the bivalent
antigen were detected by IFA, using mAb 3H5 (DEN-2 virus-specific) and
MAB8704 (DEN-4 virus-specific), in conjunction with anti-murine
IgG-rhodamine conjugate [42]. Expression of the transgene was also detected in infected, [35S]-radiolabeled cells by immunoprecipitation, using the 3H5 mAb as described previously [42].

Mouse immunization

Balb/c mice (4–6 weeks old; 5 animals per group) were immunized
using a rAd prime/plasmid boost regimen. The control group was injected
intraperitoneally (i.p) with 108 PFU of rAd-C vector (in 200 μl of 1× PBS), followed 15 days later by three intradermal (i.d) doses, again 15 days apart, of 50 μg plasmid pVAX1 vector (in 200 μl of 1× PBS). A similar regimen was followed for the test group, which was primed i.p. with 108 PFU of rAd-Bg vector and boosted i.d. with 50 μg
plasmid pVAX-EDIII-4/2 vector. Blood was drawn 1 week after priming and
after each boost for seroanalysis. Animal experiments were performed
after approval of the Institutional Animal Ethics Committee.

Seroanalysis

Antibody titers in sera of immunized mice were determined by ELISA,
in 96-well plates, using monovalent and bivalent r-EDIII proteins or
DEN viruses as capture antigens in conjunction with anti-murine
IgG-HRPO secondary antibody conjugate/3, 3' 5, 5' tetramethyl benzidine
substrate, as previously described [43].
The presence of anti-DEN-2 and anti-DEN-4 virus antibodies in immune
sera (diluted 1:100) was also detected by IFA of DEN-2 and DEN-4
virus-infected BHK cells (0.1 PFU/cell) at 24 hours post-infection.
Virus-bound antibodies were detected using anti-mouse IgG-FITC
conjugate as done earlier [43]. Neutralizing antibody titers were determined by standard PRNT assay using LLCMK2 cells as before [42,43].
The antiserum dilution resulting in 50% reduction in plaque count (with
reference to the number of plaques generated by the virus in the
absence of antiserum), was expressed as the PRNT50 titer.

T cell assays

Spleens were harvested from mice ~7 weeks after the last
immunization and cultured in 96-well plates for monitoring
proliferative response and cytokine secretion in response to in vitro stimulation with DEN-2 and DEN-4 viruses (each at 0.03 PFU/cell). Cells were pulsed with [3H]-thymidine
overnight at the end of 96 hours, and proliferation was quantitated by
measuring the uptake of the radioisotope in a scintillation counter.
IFN-γ and IL-4 were determined in
splenocyte culture supernatants, at the indicated time points, using
murine Quantikine cytokine ELISA kits (R & D systems), as described
previously [42,45]. All assays for T cell responses were run in triplicate and values expressed as mean ± standard deviation (SD).